Evolution of Quorum Sensing in Pseudomonas aeruginosa Can Occur via Loss of Function and Regulon Modulation

Pseudomonas aeruginosa populations evolving in cystic fibrosis lungs, animal hosts, natural environments and in vitro undergo extensive genetic adaption and diversification. A common mutational target is the quorum sensing (QS) system, a three-unit regulatory system that controls the expression of virulence factors and secreted public goods. Three evolutionary scenarios have been advocated to explain selection for QS mutants: (i) disuse of the regulon, (ii) cheating through the exploitation of public goods, or (ii) modulation of the QS regulon. Here, we examine these scenarios by studying a set of 61 QS mutants from an experimental evolution study. We observed nonsynonymous mutations in all three QS systems: Las, Rhl, and Pseudomonas Quinolone Signal (PQS). The majority of the Las mutants had large deletions of the Las regulon, resulting in loss of QS function and the inability to produce QS-regulated traits, thus supporting the first or second scenarios. Conversely, phenotypic and gene expression analyses of Rhl mutants support network modulation (third scenario), as these mutants overexpressed the Las and Rhl receptors and showed an altered QS-regulated trait production profile. PQS mutants also showed patterns of regulon modulation leading to strain diversification and phenotypic tradeoffs, where the upregulation of certain QS traits is associated with the downregulation of others. Overall, our results indicate that mutations in the different QS systems lead to diverging effects on the QS trait profile in P. aeruginosa populations. These mutations might not only affect the plasticity and diversity of evolved populations but could also impact bacterial fitness and virulence in infections. IMPORTANCE Pseudomonas aeruginosa uses quorum sensing (QS), a three-unit multilayered network, to coordinate expression of traits required for growth and virulence in the context of infections. Despite its importance for bacterial fitness, the QS regulon appears to be a common mutational target during long-term adaptation of P. aeruginosa in the host, natural environments, and experimental evolutions. This raises questions of why such an important regulatory system is under selection and how mutations change the profile of QS-regulated traits. Here, we examine a set of 61 experimentally evolved QS mutants to address these questions. We found that mutations involving the master regulator, LasR, resulted in an almost complete breakdown of QS, while mutations in RhlR and PqsR resulted in modulations of the regulon, where both the regulon structure and the QS-regulated trait profile changed. Our work reveals that natural selection drives diversification in QS activity patterns in evolving populations.